ACIDES NUCLEIQUES ET POLYPEPTIDES DE METHYLTRANSFERASE

METHYLTRANSFERASE NUCLEIC ACIDS AND POLYPEPTIDES

Abrégé français

Cette invention concerne l'isolation et le séquençage de molécules d'acides nucléiques qui codent pour des polypeptides de méthyltransférase provenant du cultivar Papaver somniferum ; et leurs utilisations pour produire la noscapine et identifier les cultivars de pavot qui comprennent les gènes contenant lesdites molécules d'acides nucléiques.

Abrégé anglais

This disclosure relates to the isolation and sequencing of nucleic acid molecules that encode methyltransferase polypeptides from a Papaver somniferum cultivar; and uses in the production of noscapine and identification of poppy cultivars that include the genes that comprise said nucleic acid molecules.

Revendications

37
Claims
1. An isolated polypeptide selected from the group consisting of:
i) a methyltransferase polypeptide comprising or consisting of an
amino acid sequence of SEQ ID NO: 7: and
ii) a modified polypeptide comprising or consisting of an amino acid
sequence that is at least 95% identical to the full length amino acid
sequence of SEQ ID NO: 7 which has methyltransferase activity.
2. An isolated nucleic acid molecule that encodes a methyltransferase
polypeptide
wherein said nucleic acid molecule comprises or consists of a nucleotide
sequence
selected from the group consisting of:
i) a nucleotide sequence of SEQ ID NO: 1 or 4 and
ii) a nucleotide sequence wherein said sequence is degenerate as a
result of the genetic code to the nucleotide sequence defined in (i)
and wherein said nucleic acid molecule encodes a
methyltransferase polypeptide comprising or consisting of amino
acid sequence of SEQ ID NO: 7; and
iii) a nucleic acid molecule which shares at least 90% identity over
the full length sequence of SEQ ID NO: 1 or 4 and wherein said
nucleic acid molecule encodes a methyltransferase polypeptide.
3. The isolated nucleic acid molecule according to claim 2 wherein said
nucleic acid
molecule comprises or consists of a nucleotide sequence of SEQ ID NO: 1 or 4.
4. A vector comprising a nucleic acid molecule encoding a methyltransferase
according to claim 2 or 3 whereiri said nucleic acid molecule is operably
linked with a
nucleic acid sequence comprising a promoter sequence.
5. The vector according to claim 4 wherein said nucleic acid sequence
comprising a
promoter confers constitutive expression on said methyltransferase.
6. The vector according to claim 4 wherein said nucleic acid sequence
comprising a
promoter confers regulated expression on said methyltransferase.
7. The vector according to claim 6 wherein said regulated expression is
tissue or
developmentally regulated expression.
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8. The vector according to claim 6 wherein said regulated expression is
inducible
expression.
9. A transgenic cell transformed or transfected with the nucleic acid
molecule
according to claims 2 or 3, or with the vector according to any one of claims
4 to 8.
10. A transgenic cell according to claim 9 wherein said cell is a plant
cell.
11. The transgenic cell according to claim 10 wherein said plant cell is
from the
family Papaveraceae.
12. The transgenic cell according to claim 11 wherein said plant cell is a
Papaver
somniferum cell.
13. The transgenic cell according to claim 9 wherein said cell is a
microbial cell.
14. The transgenic cell according to claim 13 wherein said microbial cell
is a bacterial
cell.
15. The transgenic cell according to claim 13 wherein said microbial cell
is a fungal
cell or yeast cell.
16. A nucleic acid molecule comprising a transcription cassette wherein
said cassette
includes the nucleotide sequence of SEQ ID NO: 1 or 4 and further comprises at
least
one promoter operably linked to said nucleotide sequence such that both sense
and
antisense molecules are transcribed from said cassette.
17. The nucleic acid molecule according to claim 16 wherein both sense and
antisense nucleic acid molecules are transcribed from said cassette wherein
said sense
and antisense nucleic acid molecules anneal over at least part or all of their
length to
form a siRNA or shRNA.
18. The nucleic acid molecule according to claim 16 or 17 wherein said
cassette is
provided with at least two promoters to transcribe both sense and antisense
strands of
said nucleic acid molecule.
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19. The nucleic acid molecule according to any one of claims 16 to 18
wherein said
cassette comprises a nucleic acid molecule wherein said molecule comprises a
first part
linked to a second part wherein said first and second parts are complementary
over at
least part of their sequence and further wherein transcription of said nucleic
acid
molecule produces an RNA molecule which forms a double stranded region by
complementary base pairing of said first and second parts thereby forming an
shRNA.
20. The nucleic acid molecule according to any of claims 16 to 19 wherein
said
nucleic acid molecule is part of a vector for expression in a plant cell.
21. A plant cell transfected with a nucleic acid molecule according to any
one of
claims 16 to 20 wherein said cell has reduced expression of said
methyltransferase,
compared to expression of methyltransferase in a plant cell not transfected
with the
nucleic acid molecule.
22. A process for the methylation of an opiate alkaloid comprising:
i) providing a transgenic plant cell according to any one of claims 10 to
12
and wherein said plant cell catalyses the transfer of the S-methyl group of s-
adenosyl-L-methionine to scoulerine to form tetrahydrocolumbamine;and
ii) cultivating said plant cell to produce a methylated opiate alkaloid.
23. A process according to claim 22 wherein said transgenic plant, or part
thereof is
harvested.
24. The process according to claim 23 wherein plant material harvested from
said
transgenic plant is dried straw and said opiate alkaloid is extracted.
25. A process for the methylation of an opiate alkaloid comprising:
i) providing a transgenic microbial cell according to any one of
claims 13 to 15 that expresses a methyltransferase according to
claim 1 in culture with at least one opiate alkaloid;
ii) cultivating the microbial cell under conditions that methylate one or
more opiate alkaloids.
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26. A process
according to claim 25 wherein said methylated alkaloid is isolated
from the microbial cell or cell culture.
27 The
process according to claim 26 wherein said microbial cell is a bacterial cell,
a
fungal cell or a yeast cell.
28. The use
of a nucleic acid encoded by the nucleic acid molecule of SEQ ID NO: 4
or a nucleic acid molecule with at least 90% sequence identity over the full
length of the
sequence SEQ ID NO: 4 wherein said nucleic acid molecule encodes a
methyltransferase polypeptide as a means to identify the presence or absence
of a
nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 4, that
encodes
said methyltransferase in a Papaveraceae plant.
29. A method
to determine the presence or absence of a nucleic acid
comprising or consisting of the nucleic acid molecule sequence of SEQ ID NO: 4
in a
Papaveraceae variety comprising:
i) obtaining a sample from a Papaveraceae plant;
ii) extracting genomic DNA from the plant; and
iii) analyzing the genomic DNA for the presence of a gene comprising or
consisting of a nucleotide molecule sequence with SEQ ID NO: 4.
30. The use
of the nucleic acid molecule of SEQ ID NO: 4 or a nucleic acid molecule
with at least 90% sequence identity over the full length sequence of SEQ ID
NO: 4
wherein said nucleic acid molecule encodes a methyltransferase polypeptide, to
identify
a locus with expression or activity of the methyltransferase polypeptide.
31. A method
to produce a Papaveraceae plant variety that has expression of a
methyltransferase comprising the steps of:
i) mutagenesis of wild-type seed from a plant that does express said
methyltransferase;
ii) cultivation of the seed in i) to produce first and subsequent
generations of plants;
iii) obtaining seed from the first generation plant and subsequent
generations of plants ;
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iv) determining if the seed from said first and subsequent generations
of plants has altered expression of said methyltransferase in
comparison to the expression of the methyltransferase in the plant
from the wild type seed;
v) obtaining a sample and analysing the nucleic acid sequence of a
nucleic acid molecule selected from the group consisting of:
a) a nucleic acid molecule comprising a nucleotide
molecule sequence with SEQ ID NO: 4;
b) a nucleic acid molecule with at least 90% sequence
identity over the full length of SEQ ID NO: 4 and
wherein said nucleic acid molecule encodes a
methyltransferase polypeptide.
32. The method according to claim 31 comprising further the step of
comparing the
nucleotide sequence of the nucleic acid molecule in said sample under v) to a
nucleotide
sequence of a nucleic acid molecule of the original wild-type plant.
33. The method according to claim 32 wherein said nucleic acid molecule is
analysed
by a method comprising the steps of:
i) extracting nucleic acid from said mutated plants;
ii) amplification of a part of said nucleic acid molecule by a polymerase
chain reaction;
iii) forming a preparation comprising the amplified nucleic acid and nucleic
acid extracted from wild-type seed to form heteroduplex nucleic acid;
iv) incubating said preparation with a single stranded nuclease that cuts at a
region of heteroduplex nucleic acid to identify the mismatch in said
heteroduplex; and
v) determining the site of the mismatch in said nucleic acid heteroduplex.
34. A viral vector comprising all or part of a nucleic acid molecule
according to claim
2 or 3.
35. The use of a viral vector according to claim 34 in viral induced gene
silencing in a
plant.
36. Use according to claim 35 wherein said plant is from the family
Papaveraceae.
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Description

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Methvltransf erase Nucleic Acids and Polvpeptides
Introduction
This disclosure relates to the isolation and sequencing of nucleic acid
molecules that
encode methyltransferase polypeptides from a Papaver somniferum cultivar,
[poppy
plant]; transgenic cells transformed with said nucleic acid molecules,
sequence variants
of the genes; the use of said genes/proteins in the production of noscapine
and the use
of the genes as markers of poppy plants that synthesize noscapine.
Background
The opium poppy P. somniferum is the plant from which opium is extracted. The
opium
poppy is the only commercially exploited poppy of the family Papa veraceae and
is the
principal source of natural opiates. The opium is extracted from latex
harvested from the
green seed pods. A further source of opiate alkaloids is the poppy straw which
is the
dried mature plant. P. somniferum is a source of clinically useful opiate
alkaloids such
as morphine, codeine, thebaine, noscapine [also known as narcotine] and
papaverine.
The clinical application of these opiate alkaloids and their derivates is
broad having use
as analgesics, cough suppressants and anti-spasmodics. Although not used as a
pharmacological agent in its own right, thebaine is a particularly useful
opiate which can
be converted into a range of compounds such as hydrocodone, oxycodone,
oxymorphone, nalbuphine naltrexone, buprenorphine and etorphine. These
intermediates also have broad pharmaceutical applications. For example,
oxycodone,
oxymorphone and etorphine are widely used as an analgesic for moderate to
severe
pain and are often combined with other analgesics such as ibuprofen.
Buprenorphine is
used in the treatment of heroin addiction and chronic pain. Naltrexone is used
in the
treatment of alcohol and opiate addiction.
The use of thebaine in the production of these compounds is limited since
thebaine is a
minor component of the opiates extracted from poppy straw only accounting for
approximately 0.5-2% of the opium extracted from dry straw. Mutant varieties
of P.
somniferum have been developed that can obtain thebaine and oripavine content
of at
least 50% by weight of the alkaloid combination of morphine, codeine, thebaine
and
oripavine; see W098/02033. Alternative means to enhance the production of
thebaine
include the spraying of poppy plants with growth regulatory chemicals which
inhibit

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alkaloid biosynthetic pathways to enhance the production of thebaine and other
opiate
alkaloids.
This disclosure relates to molecular analyses of gene expression in poppy
cultivars that
.. produce noscapine. Noscapine does not have significant analgesic properties
but is
used as a cough suppressant and is being investigated as an anti-cancer agent
and in
the treatment of stroke patients.
We have surprisingly found that certain varieties of poppy cultivars have
genes that are
unique to those cultivars that produce noscapine. We have cloned three genes
that have
homology to methyltransf erases.
The first methyltransferase, PSMT1, exhibits sequence similarity to S-Adenosyl-
L-
Methionine:Scoulerine-9-0-Methyltransf erase from Coptis japonica (Accession:
Q39522.1, 61% identical) and from Thalictrum flavum (Accession: AAU20770.1,
59%
identical). The protein from Coptis japonica has been characterised and shown
to
catalyse the transfer of the S-methyl group of S-adenosyl-L-methionine to the
9-hydroxyl
group of scoulerine to form tetrahydrocolumbamine (Sato et al. (1993)
Phytochem.
32:659-664) which, in turn, serves as the precursor for the synthesis of most
protoberberine alkaloids.
The second methyltransferase, PSMT2, exhibits sequence similarity to S-
Adenosyl-L-
Methionine:Norcoclaurine-6-0-Methyltransferase from Coptis japonica
(Accession:
Q9LEL6, 42% identical) which has been shown to catalyse the transfer of the S-
methyl
group of S-adenosyl-L-methionine to the 6-hydroxyl group of (S)-Norcoclaurine
to form
(S)-Coclaurine (Morishige et al. (2000) J. Biol. Chem. 275(30): 23398-23405).
The third methyltransferase, PSMT3, exhibits sequence similarity to S-Adenosyl-
L-
Methionine:Norcoclaurine-6-0-Methyltransferase from Papaver somniferum
(Accession:
AAQ01669, 80% identical) and Papaver bracteatum (Accession: AC090232, 80%
identical). The protein from Papaver somniferum has been characterised and
shown to
catalyse the transfer of the S-methyl group of S-adenosyl-L-methionine to to
catalyse the
transfer of the S-methyl group of S-adenosyl-L-methionine to the 6-hydroxyl
group of
(S)-Norcoclaurine to form (S)-Coclaurine (Ounaroon et al. (2003) 36:808-819).

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Statements of Invention
According to an aspect of the invention there is provided an isolated nucleic
acid
molecule that encodes a methyltransferase polypeptide wherein said nucleic
acid
molecule comprises or consists of a nucleotide sequence selected from the
group
consisting of:
i) a nucleotide sequence as represented by the sequence in Figure la, lb
lc, 3a, 3b or 3c;
ii) a nucleotide sequence wherein said sequence is degenerate as a result
of the genetic code to the nucleotide sequence defined in (i);
iii) a nucleic acid molecule the complementary strand of which hybridizes
under stringent hybridization conditions to the sequence in Figure la, 1 b,
lc, 3a, 3b or 3c wherein said nucleic acid molecule encodes a
methyltransferase polypeptide;
iv) a nucleotide
sequence that encodes a polypeptide comprising an amino
acid sequence as represented in Figure 4a, 4b or 4c;
v) a nucleotide sequence that encodes a polypeptide comprising an amino
acid sequence wherein said amino acid sequence is modified by addition
deletion or substitution of at least one amino acid residue as represented
in iv) above and which has retained or enhanced methy transferase
activity.
Hybridization of a nucleic acid molecule occurs when two complementary nucleic
acid
molecules undergo an amount of hydrogen bonding to each other. The stringency
of
hybridization can vary according to the environmental conditions surrounding
the nucleic
acids, the nature of the hybridization method, and the composition and length
of the
nucleic acid molecules used. Calculations regarding hybridization conditions
required for
attaining particular degrees of stringency are discussed in Sambrook et al.,
Molecular
Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and
Molecular
Biology¨Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier,
New York,
1993). The Tm is the temperature at which 50% of a given strand of a nucleic
acid
molecule is hybridized to its complementary strand. The following is an
exemplary set of
hybridization conditions and is not limiting:
Very High Stringency (allows sequences that share at least 90% identity to
hybridize)

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Hybridization: 5x SSC at 65 C for 16 hours
Wash twice: 2x SSC at room temperature (RT) for 15 minutes each
Wash twice: 0.5x SSC at 65 C for 20 minutes each
High Stringency (allows sequences that share at least 80% identity to
hybridize)
Hybridization: 5x-6x SSC at 65 C-70 C for 16-20 hours
Wash twice: 2x SSC at RT for 5-20 minutes each
Wash twice: lx SSC at 55 C-70 C for 30 minutes each
Low Stringency (allows sequences that share at least 50% identity to
hybridize)
Hybridization: 6x SSC at RT to 55 C for 16-20 hours
Wash at least twice: 2x-3x SSC at RT to 55 C for 20-30 minutes each.
In a preferred embodiment of the invention said nucleic acid molecule
comprises or
consists of a nucleotide sequence as represented in Figure la, lb, lc, 3a, 3b
or 3c.
According to a further aspect of the invention there is provided an isolated
polypeptide
selected from the group consisting of:
i) a polypeptide comprising or consisting of an amino acid
sequence as
represented in Figure 4a, 4b or 4c; or
ii) a modified polypeptide comprising or consisting of a modified amino
acid
sequence wherein said polypeptide is modified by addition deletion or
substitution of at least one amino acid residue of the sequence presented
in Figure 4a, 4b or 4c and which has retained or enhanced
methyltransferase activity.
A modified polypeptide as herein disclosed may differ in amino acid sequence
by one or
more substitutions, additions, deletions, truncations that may be present in
any
combination. Among preferred variants are those that vary from a reference
polypeptide
by conservative amino acid substitutions. Such substitutions are those that
substitute a
given amino acid by another amino acid of like characteristics. The following
non-limiting
list of amino acids are considered conservative replacements (similar): a)
alanine,
serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and
glutamine d)
arginine and lysine; e) isoleucine, leucine, methionine and valine and f)
phenylalanine,
tyrosine and tryptophan. Most highly preferred are variants that retain or
enhance the
same biological function and activity as the reference polypeptide from which
it varies.

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In one embodiment, the variant polypeptides have at least 43% identity, more
preferably
at least 62% identity, even more preferably at least 70% identity, still more
preferably at
5 least 75%, 80%, 85%, 90%, 95% identity, and most preferably at least 99%
identity with
the full length amino acid sequence illustrated herein.
According to a further aspect of the invention there is provided a vector
comprising a
nucleic acid molecule encoding a methyltransferase according to the invention
wherein
said nucleic acid molecule is operably linked with a nucleic acid sequence
comprising a
promoter sequence.
In a preferred embodiment of the invention said nucleic acid sequence
comprising a
promoter confers constitutive expression on said methyltransf erase.
In an alternative preferred embodiment of the invention said nucleic acid
sequence
comprising a promoter confers regulated expression on said methyltransferase.
In a preferred embodiment of the invention said regulated expression is tissue
or
developmentally regulated expression.
In a further alternative embodiment of the invention said regulated expression
is
inducible expression.
.. In an alternative embodiment of the invention a vector including nucleic
acid according to
the invention need not include a promoter or other regulatory sequence,
particularly if
the vector is to be used to introduce the nucleic acid into cells for
recombination into the
gene.
Preferably the nucleic acid in the vector is under the control of, and
operably linked to,
an appropriate promoter or other regulatory elements for transcription in a
host cell such
as a microbial, (e.g. bacterial, yeast), or plant cell. The vector may be a bi-
functional
expression vector which functions in multiple hosts. In the case of
methyltransferase
genomic DNA this may contain its own promoter or other regulatory elements and
in the
case of cDNA this may be under the control of an appropriate promoter or other
regulatory elements for expression in the host cell.

6
By "promoter" is meant a nucleotide sequence upstream from the transcriptional
initiation site and which contains all the regulatory regions required for
transcription.
Suitable promoters include constitutive, tissue-specific, inducible,
developmental or other
promoters for expression in plant cells comprised in plants depending on
design. Such
promoters include viral, fungal, bacterial, animal and plant-derived promoters
capable of
functioning in plant cells.
Constitutive promoters include, for example CaMV 35S promoter (Odell et al.
(1985)
.. Nature 313: 9810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-
171); ubiquitin
(Christian et al. (1989) Plant Mol. Biol. 18: 675-689); pEMU (Last et at.
(1991) Theor
Appl. Genet. 81: 581-588); MAS (Velten et at. (1984) EMBO J. 3: 2723-2730);
ALS
promoter (U.S. Application Serie! No. 08/409,297), and the like. Other
constitutive
promoters include those in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121;
5,569,597; 5,466,785; 5,399,680, 5,268,463; and 5,608,142.
Chemical-regulated promoters can be used to modulate the expression of a gene
in a
plant through the application of an exogenous chemical regulator. Depending
upon the
objective, the promoter may be a chemical-inducible promoter, where
application of the
chemical induced gene expression, or a chemical-repressible promoter, where
application of the chemical represses gene expression. Chemical-inducible
promoters
are known in the art and include, but are not limited to, the maize In2-2
promoter, which
is activated by benzenesulfonamide herbicide safeners, the maize GST promoter,
which
is activated by hydrophobic electrophilic compounds that are used as pre-
emergent
herbicides, and the tobacco PR-la promoter, which is activated by salicylic
acid. Other
chemical-regulated promoters of interest include steroid-responsive promoters
(see, for
example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc.
Natl. Acad.
Sci. USA 88: 10421-10425 and McNellis et at. (1998) Plant J. 14(2): 247-257)
and
tetracycline-inducible and tetracycline-repressible promoters (see, for
example, Gatz et
at. (1991) Mol. Gen. Genet. 227: 229-237, and US Patent Nos. 5,814,618 and
5,789,156.
Where enhanced expression in particular tissues is desired, tissue-specific
promoters
.. can be utilised. Tissue-specific promoters include those described by
Yamamoto et al.
(1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol.
38(7): 792-
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803; Hansen et al. (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al.
(1997)
Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3):
1331-1341;
Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascni et al.
(1996) Plant
Physiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):
773-778;
Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant
Mol. Biol.
23(6): 1129-1138; Mutsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90 (20):
9586-
9590; and Guevara-Garcia et al (1993) Plant J. 4(3): 495-50.
"Operably linked" means joined as part of the same nucleic acid molecule,
suitably
positioned and oriented for transcription to be initiated from the promoter.
DNA operably
linked to a promoter is "under transcriptional initiation regulation" of the
promoter. In a
preferred aspect, the promoter is a tissue specific promoter, an inducible
promoter or a
developmentally regulated promoter.
Particular of interest in the present context are nucleic acid constructs
which operate as
plant vectors. Specific procedures and vectors previously used with wide
success in
plants are described by Guerineau and Mullineaux (1993) (Plant transformation
and
expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford,
BIOS
Scientific Publishers, pp 121-148. Suitable vectors may include plant viral-
derived
vectors (see e.g. EP194809).
If desired, selectable genetic markers may be included in the construct, such
as those
that confer selectable phenotypes such as resistance to herbicides (e.g.
kanamycin,
hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin,
spectinomycin,
imidazolinones and glyphosate).
According to a further aspect of the invention there is provided a transgenic
cell
transformed or transfected with a nucleic acid molecule or vector according to
the
invention.
In a preferred embodiment of the invention said cell is a plant cell.
In a preferred embodiment of the invention said plant cell is from the family
Papa veraceae.
In a preferred embodiment of the invention said plant cell is a Papaver
somniferum cell.

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According to a further aspect of the invention there is provided a plant
comprising a plant
cell according to the invention.
In a preferred embodiment of the invention said plant is from the family
Papaveraceae;
preferably Papa ver somniferum.
In an alternative preferred embodiment of the invention said cell is a
microbial cell;
preferably a bacterial or fungal cell [e.g. yeast, Saccharomyces cerevisae].
According to a further aspect of the invention there is provided a nucleic
acid molecule
comprising a transcription cassette wherein said cassette includes a
nucleotide
sequence designed with reference to Figure la, 1 b, lc, 3a, 3b or 3c and is
adapted for
expression by provision of at least one promoter operably linked to said
nucleotide
sequence such that both sense and antisense molecules are transcribed from
said
cassette.
In a preferred embodiment of the invention said cassette is adapted such that
both
sense and antisense nucleic acid molecules are transcribed from said cassette
wherein
said sense and antisense nucleic acid molecules are adapted to anneal over at
least part
or all of their length to form a siRNA or shRNA.
In a preferred embodiment of the invention said cassette is provided with at
least two
promoters adapted to transcribe both sense and antisense strands of said
nucleic acid
molecule.
In an alternative preferred embodiment of the invention said cassette
comprises a
nucleic acid molecule wherein said molecule comprises a first part linked to a
second
part wherein said first and second parts are complementary over at least part
of their
sequence and further wherein transcription of said nucleic acid molecule
produces an
RNA molecule which forms a double stranded region by complementary base
pairing of
said first and second parts thereby forming an shRNA.
A technique to specifically ablate gene function is through the introduction
of double
stranded RNA, also referred to as small inhibitory/interfering RNA (siRNA) or
short
hairpin RNA [shRNA], into a cell which results in the destruction of mRNA
complementary to the sequence included in the siRNA/shRNA molecule. The siRNA

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molecule comprises two complementary strands of RNA (a sense strand and an
antisense strand) annealed to each other to form a double stranded RNA
molecule. The
siRNA molecule is typically derived from exons of the gene which is to be
ablated. The
mechanism of RNA interference is being elucidated. Many organisms respond to
the
presence of double stranded RNA by activating a cascade that leads to the
formation of
siRNA. The presence of double stranded RNA activates a protein complex
comprising
RNase III which processes the double stranded RNA into smaller fragments
(siRNAs,
approximately 21-29 nucleotides in length) which become part of a
ribonucleoprotein
complex. The siRNA acts as a guide for the RNase complex to cleave mRNA
complementary to the antisense strand of the siRNA thereby resulting in
destruction of
the mRNA.
In a preferred embodiment of the invention said nucleic acid molecule is part
of a vector
adapted for expression in a plant cell.
According to a further aspect of the invention there is provided a plant cell
transfected
with a nucleic acid molecule or vector according to the invention wherein said
cell has
reduced expression of said methyltransferase.
According to an aspect of the invention there is provided a process for the
methylation of
an opiate alkaloid comprising:
i) providing a transgenic plant cell according to the invention;
ii) cultivating said plant cell to produce a transgenic plant; and
optionally
i) harvesting said transgenic plant, or part thereof.
In a preferred method of the invention said harvested plant material is dried
straw and
said opiate alkaloid is extracted.
According to an alternative aspect of the invention there is provided a
process for the
methylation of an opiate alkaloid comprising:
i) providing a transgenic microbial cell according to the invention that
expresses a methy transf erase according to the invention in culture with
at least one opiate alkaloid;
ii) cultivating the microbial cell under conditions that methylate one or
more
opiate alkaloids; and optionally
iii) isolating said methylated alkaloid from the microbial cell or cell
culture.

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In a preferred method of the invention said microbial cell is a bacterial cell
or
fungal/yeast cell.
If microbial cells are used as organisms in the process according to the
invention they
5 are grown or cultured in the manner with which the skilled worker is
familiar, depending
on the host organism. As a rule, microorganisms are grown in a liquid medium
comprising a carbon source, usually in the form of sugars, a nitrogen source,
usually in
the form of organic nitrogen sources such as yeast extract or salts such as
ammonium
sulfate, trace elements such as salts of iron, manganese and magnesium and, if
10 appropriate, vitamins, at temperatures of between 0 C and 100 C,
preferably between
10 C and 60 C, while gassing in oxygen.
The pH of the liquid medium can either be kept constant, that is to say
regulated during
the culturing period, or not. The cultures can be grown batchwise, semi-
batchwise or
continuously. Nutrients can be provided at the beginning of the fermentation
or fed in
semi-continuously or continuously. The methylated opiate alkaloids produced
can be
isolated from the organisms as described above by processes known to the
skilled
worker, for example by extraction, distillation, crystallization, if
appropriate precipitation
with salt, and/or chromatography. To this end, the organisms can
advantageously be
disrupted beforehand. In this process, the pH value is advantageously kept
between pH
4 and 12, preferably between pH 6 and 9, especially preferably between pH 7
and 8.
The culture medium to be used must suitably meet the requirements of the
strains in
question. Descriptions of culture media for various microorganisms can be
found in the
textbook "Manual of Methods for General Bacteriology" of the American Society
for
Bacteriology (Washington D.C., USA, 1981).
As described above, these media which can be employed in accordance with the
invention usually comprise one or more carbon sources, nitrogen sources,
inorganic
salts, vitamins and/or trace elements.
Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
Examples
of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose,
ribulose,
lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be
added to
the media via complex compounds such as molasses or other by-products from
sugar
refining. The addition of mixtures of a variety of carbon sources may also be
advantageous. Other possible carbon sources are oils and fats such as, for
example,

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soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as,
for example,
palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols
such as, for
example, glycerol, methanol and/or ethanol, and/or organic acids such as, for
example,
acetic acid and/or lactic acid.
Nitrogen sources are usually organic or inorganic nitrogen compounds or
materials
comprising these compounds. Examples of nitrogen sources comprise ammonia in
liquid
or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride,
ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea,
amino
acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya
protein,
yeast extract, meat extract and others. The nitrogen sources can be used
individually or
as a mixture.
Inorganic salt compounds which may be present in the media comprise the
chloride,
phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt,
molybdenum,
potassium, manganese, zinc, copper and iron.
Inorganic sulfur-containing compounds such as, for example, sulfates,
sulfites,
dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur
compounds such
as mercaptans and thiols may be used as sources of sulfur for the production
of sulfur-
containing fine chemicals, in particular of methionine.
Phosphoric acid, potassium dihydrogenphosphate or dipotassium
hydrogenphosphate or
the corresponding sodium-containing salts may be used as sources of
phosphorus.
Chelating agents may be added to the medium in order to keep the metal ions in
solution. Particularly suitable chelating agents comprise dihydroxyphenols
such as
catechol or protocatechuate and organic acids such as citric acid.
The fermentation media used according to the invention for culturing
microorganisms
usually also comprise other growth factors such as vitamins or growth
promoters, which
include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic
acid, panthothenate
and pyridoxine. Growth factors and salts are frequently derived from complex
media
components such as yeast extract, molasses, cornsteep liquor and the like. It
is
moreover possible to add suitable precursors to the culture medium. The exact
composition of the media compounds heavily depends on the particular
experiment and
is decided upon individually for each specific case. Information on the
optimization of
media can be found in the textbook "Applied Microbiol. Physiology, A Practical
Approach" (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73,
ISBN 0 19

CA 02803102 2012-12-18
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WO 2011/161431 PCT/GB2011/051121
963577 3). Growth media can also be obtained from commercial suppliers, for
example
Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
All media components are sterilized, either by heat (20 min at 1.5 bar and 121
C) or by
filter sterilization. The components may be sterilized either together or, if
required,
separately. All media components may be present at the start of the
cultivation or added
continuously or batchwise, as desired.
The culture temperature is normally between 15 C and 45 C, preferably at from
25 C to
40 C, and may be kept constant or may be altered during the experiment. The pH
of the
medium should be in the range from 5 to 8.5, preferably around 7Ø The pH for
cultivation can be controlled during cultivation by adding basic compounds
such as
sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic
compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled
by
employing antifoams such as, for example, fatty acid polyglycol esters. To
maintain the
stability of plasmids it is possible to add to the medium suitable substances
having a
selective effect, for example antibiotics. Aerobic conditions are maintained
by introducing
oxygen or oxygen-containing gas mixtures such as, for example, ambient air
into the
culture. The temperature of the culture is normally 20 C to 45 C and
preferably 25 C to
40 C. The culture is continued until formation of the desired product is at a
maximum.
This aim is normally achieved within 10 to 160 hours.
The fermentation broth can then be processed further. The biomass may,
according to
requirement, be removed completely or partially from the fermentation broth by
separation methods such as, for example, centrifugation, filtration, decanting
or a
combination of these methods or be left completely in said broth. It is
advantageous to
process the biomass after its separation.
However, the fermentation broth can also be thickened or concentrated without
separating the cells, using known methods such as, for example, with the aid
of a rotary
evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis
or by
nanofiltration. Finally, this concentrated fermentation broth can be processed
to obtain
the opiate alkaloids present therein.
According to a further aspect of the invention there is provided the use of a
gene
encoded by a nucleic acid molecule as represented by the nucleic acid sequence
in
Figure 3a, 3b or 3c, or a nucleic acid molecule that hybridizes under
stringent
hybridization conditions to the nucleotide sequence in Figure 3a, 3b or 3c and
encodes a

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PCT/GB2011/051121
polypeptide with methyltransferase activity as a means to identify the
presence or
absence of a gene that encodes said methyltransf erase in a Papa veraceae
plant.
According to a further aspect of the invention there is provided a method to
determine
the presence or absence of a gene according to the invention in a Papa
veraceae variety
comprising:
i) obtaining a sample from a Papaveraceae plant;
ii) extracting genomic DNA from the plant; and
iii) analyzing the genomic DNA for the presence of a gene comprising or
consisting of a nucleotide sequence as represented in Figure 3a, 3b or 3c.
Methods to analyze genomic DNA are well known in the art. For example,
polymerase
chain reaction methods using sequence specific oligonucleotide primers to
amplify
specific regions of the gene according to the invention. The extraction,
isolation and
restriction analysis using sequence specific restriction endonucleases
followed by
separation and Southern blotting to analyze genomic structure have been
established for
over thirty years. The analysis may be directed to intron or exon structure or
upstream or
downstream regions of the gene; e.g. promoter regions.
According to a further aspect of the invention there is provided the use of a
gene
encoded by a nucleic acid molecule as represented by the nucleic acid sequence
in
Figure 3a, 3b or 3c, or a nucleic acid molecule that hybridizes under
stringent
hybridization conditions to the nucleotide sequence in Figure 3a, 3b or 3c and
encodes a
polypeptide with methyltransferase activity as a means to identify a locus
wherein said
locus is associated with altered expression or activity of said methyltransf
erase.
Mutagenesis as a means to induce phenotypic changes in organisms is well known
in
the art and includes but is not limited to the use of mutagenic agents such as
chemical
mutagens [e.g. base analogues, deaminating agents, DNA intercalating agents,
alkylating agents, transposons, bromine, sodium azide] and physical mutagens
[e.g.
ionizing radiation, psoralen exposure combined with UV irradiation].
According to a further aspect of the invention there is provided a method to
produce a
Papa veraceae plant variety that has altered expression of a methyltransferase
according
to the invention comprising the steps of:

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i)
mutagenesis of wild-type seed from a plant that does express said
methyltransferase;
ii) cultivation of the seed in i) to produce first and subsequent
generations of
plants;
iii) obtaining seed
from the first generation plant and subsequent generations
of plants ;
iv) determining if the seed from said first and subsequent generations of
plants has altered nucleotide sequence and/or altered expression of said
methyltransferase;
v) obtaining a sample and analysing the nucleic acid sequence of a nucleic
acid molecule selected from the group consisting of:
a) a nucleic acid molecule comprising a nucleotide sequence as
represented in Figure 3a, 3b or 3c;
b) a nucleic acid molecule that hybridises to the nucleic acid
molecule in a) under stringent hybridisation conditions and
that encodes a polypeptide with methyltransferase activity;
and optionally
vi) comparing the nucleotide sequence of the nucleic acid molecule in said
sample to a nucleotide sequence of a nucleic acid molecule of the original
wild-type plant.
In a preferred method of the invention said nucleic acid molecule is analysed
by a
method comprising the steps of:
i) extracting nucleic acid from said mutated plants;
ii) amplification of a part of said nucleic acid molecule by a polymerase
chain reaction;
iii) forming a preparation comprising the amplified nucleic acid and nucleic
acid extracted from wild-type seed to form heteroduplex nucleic acid;
iv) incubating said preparation with a single stranded nuclease that cuts at a
region of heteroduplex nucleic acid to identify the mismatch in said
heteroduplex; and
v) determining the site of the mismatch in said nucleic acid heteroduplex.
In a preferred method of the invention said Papaveraceae plant variety has
enhanced
methyltransferase expression and/or activity.

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According to a further aspect of the invention there is provided a plant
obtained by the
method according to the invention.
5 According to an aspect of the invention there is provided a plant wherein
said plant
comprises a viral vector that includes all or part of a gene comprising a
nucleic acid
molecule according to the invention.
In a preferred embodiment of the invention said gene is encoded by a nucleic
acid
10 molecule comprising a nucleic acid sequence selected from the group
consisting of:
i) a nucleic acid molecule comprising a nucleotide sequence as represented
in Figure la, lb or 1 c ;
ii) a nucleic acid molecule comprising a nucleotide sequence that
hybridises
under stringent hybridisation conditions to a nucleic acid molecule in (i)
15 and which encodes a methytransferase polypeptide;
iii) a nucleic acid molecule that encodes a variant polypeptide that varies
from a polypeptide comprising the amino acid sequence as represented in
Figure 4a, 4b or 4c.
In a preferred embodiment of the invention said nucleic acid molecule
comprises or
consists of a nucleotide sequence as represented in Figure la.
In a preferred embodiment of the invention said nucleic acid molecule
comprises or
consists of a nucleotide sequence as represented in Figure lb.
In a preferred embodiment of the invention said nucleic acid molecule
comprises or
consists of a nucleotide sequence as represented in Figure lc
In a preferred embodiment of the invention said nucleic acid molecule consists
of a
nucleotide sequence as represented in Figure 14.
In an alternative preferred embodiment of the invention said nucleic acid
molecule
consists of a nucleotide sequence as represented in Figure 15.

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According to a further aspect of the invention there is provided a viral
vector comprising
all or part of a nucleic acid molecule according to the invention.
According to an aspect of the invention there is provided the use of a viral
vector
according to the invention in viral induced gene silencing in a plant.
In a preferred embodiment of the invention said plant is from the family Papa
veraceae.
Virus induced gene silencing [VIGS] is known in the art and exploits a RNA
mediated
antiviral defence mechanism. Plants that are infected with an unmodified virus
induces a
mechanism that specifically targets the viral genome. However, viral vectors
which are
engineered to include nucleic acid molecules derived from host plant genes
also induce
specific inhibition of viral vector expression and additionally target host
mRNA. This
allows gene specific gene silencing without genetic modification of the plant
genome and
is essentially a non-transgenic modification.
Throughout the description and claims of this specification, the words
"comprise" and
"contain" and variations of the words, for example "comprising" and
"comprises", means
"including but not limited to", and is not intended to (and does not) exclude
other
moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular
encompasses
the plural unless the context otherwise requires. In particular, where the
indefinite article
is used, the specification is to be understood as contemplating plurality as
well as
singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups
described
in conjunction with a particular aspect, embodiment or example of the
invention are to be
understood to be applicable to any other aspect, embodiment or example
described
herein unless incompatible therewith.
An embodiment of the invention will now be described by example only and with
reference to the following figures:
Figure la is the nucleotide sequence of a cDNA that encodes PSMT1; Figure lb
is the
nucleotide sequence of a cDNA that encodes PSMT2; Figure lc is the nucleotide
sequence of a cDNA that encodes PSMT3

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Figure 2: Expression of the P. somniferum SAM-Scoulerine-9-0-methyltransferase
gene
(PSMT1). The 16 EST libraries were generated by pyrosequencing using cDNA
libraries
prepared from stems (S) and capsules (C) at two developmental stages 'early
harvest'
(EH, 1-3 days after petals had fallen off) and late-harvest' (LH, 4-6 days
after petals had
fallen off) from each of the four P. somniferum cultivars: GSK MORHINE CVS1,
GSK
MORPHINE CVS2, GSK NOSAPINE CVS1 and GSK THEBAINE CVS1;
Figure 3a is the genomic nucleotide sequence of PSMT1; Figure 3b is the
genomic
nucleotide sequence of PSMT2: Figure 3c is the genomic nucleotide sequence of
PSMT3;
Figure 4a is the deduced amino acid sequence of PSMT1; Figure 4b is the
deduced
amino acid sequence of PSMT2; Figure 4c is the deduced amino acid sequence of
PSMT3;
Figure 5: PCR-amplification of PsSOMT1 from genomic DNA of the four Papaver
somniferum cultivars GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK
NOSCAPINE CVS1, GSK THEBAINE CVS1;
Figure 6a is a tabular representation of the segregation of PSMT1 gene in an
F2
mapping population derived from a parental cross of cultivars GSK NOSCAPINE
CVS1
and GSK THEBAINE CVS1 along with the co-segregation of the PSMT1 gene and
noscapine accumulation in individual F2 plants; Figure 6b is the equivalent
tabular
representation of the segregation of the PSMT2 gene. The PSMT2 genotyping
assay
failed (as indicated by the failure to amplify the internal positive control)
on six DNA
samples from F2 plants that were positive for PSMT1 and PSMT3 (indicated by
asterisks); Figure 6c is the equivalent tabular representation of the
segregation of the
PSMT3 gene;
Figure 7 illustrates a typical UPLC chromatogram for standard solution;
Figure 8 illustrates a typical UPLC chromatogram for a noscapine containing
poppy
variety;

18
Figure 9: Expression of the P. somniferum methyltransferase gene (PSMT2). The
16 EST
libraries were generated by pyrosequencing using cDNA libraries prepared from
stems (S)
and capsules (C) at two developmental stages 'early harvest' (EH, 1-3 days
after petals had
fallen off) and 'late-harvest' (LH, 4-6 days after petals had fallen off) from
each of the four
P. somniferum cultivars: GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK
NOSCAPINE CVS1, GSK THEBAINE CVS1;
Figure 10 shows the results of a PCR-amplification of PSMT2 from genomic DNA
of the four
Papaver somniferum cultivars GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK
NOSCAPINE CVS1, GSK THEBAINE CVS1;
Figure 11 Expression of the P. somniferum methyltransferase gene ( PSMT3). The
16 EST
libraries were generated by pyrosequencing using cDNA libraries prepared from
stems (S)
and capsules (C) at two developmental stages 'early harvest' (EH, 1-3 days
after petals had
.. fallen off) and 'late-harvest' (LH, 4-6 days after petals had fallen off)
from each of the four
P. somniferum cultivars: GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK
NOSCAPINE CVS1, GSK THEBAINE CVS1;
Figure 12 shows the results of a PCR-amplification of PSMT3 from genomic DNA
of the four
Papaver somniferum cultivars GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK
NOSCAPINE CVS1, GSK THEBAINE CVS1;
Figure 13 is the 622 bases long part of the PHYTOENE DESATURASE gene sequence
amplified from cDNA of GSK NOSCAPINE CVS1. The 129-bases long sequence stretch
used to silence the PHYTOENE DESATURASE gene is underlined;
Figure 14 is the part of the cDNA sequence used to silence PSMT1;
Figure 15 is the part of the cDNA sequence used to silence PSMT2;
Figure 16A and 16B shows the normalised peak area of scoulerine in the UPLC
chromatograms obtained from latex (16A) and mature capsules (16B) of plants
that
displayed the photo-bleaching phenotype after infection with the silencing
constructs pTRV2-
PDS-PSMT1, pTRV2-PDS-PSMT2 or pTRV2-PDS, respectively. The scoulerine peak
area
obtained from uninfected plants is shown as well. 21-30 plants were analysed
per construct;
and
Date Recue/Date Received 2020-06-18

19
Figure 17A and 17B shows the normalised peak area of putative narcotoline in
the UPLC
chromatograms obtained from latex (17A) and mature capsules (17B) of plants
that
displayed the photo-bleaching phenotype after infection with the silencing
constructs pTRV2-
PDS-PSMT1, pTRV2-PDS-PSMT2 or pTRV2-PDS, respectively. The putative
narcotoline
peak area obtained from uninfected plants is shown as well. 21-30 plants were
analysed per
construct.
Tables
Table 1: Sequences of forward and reverse primers used to amplify PS
METHYL TRANSFERASEs from genomic DNA
Methyltransferase Primer name Oligonucleotide sequences (5'¨ to 3'-)
gene
PSMT1_F2 CTCTAAAATGCCAAACGCG
PSMT1_F3 CGTGTTGTTTGGTCCCTCG
PSMT1_F4 GATTCCCGATTTACTCCTGATGG
PSMT1_F5 TGCCTCATGTTATTTCTGTTGCC
PSMT1_F6 CACACCAAACTTGATCATTGTC
PSMT1 PSMT1_R1 GCACACTGTCTTTTTCTTCCACC
PSMT1_R2 GACCCTTTGGGACTTCCTCG
PSMT1_R3 AAATCGTTCGCTCTTTACCGC
PSMT1 R4 AACACAAAATACGATTACTTACTTTTGTC
_
C
PSMT1_R5 GCATGAAATGGATGTAGTTATCTTGG
PSMT2_F1 ATTGTTGATATTGAATCAGAAACTTTC
PSMT2_F2 TCAATACCAGTACTGTTAGTTTCCG
PSMT2_F4 GCAACTGTTTCATTAACAGGCACATCC
PSMT2
PSMT2 ¨F5 ATTGATGTCGGTGGTGGTCACG
PSMT2_R3 CCACATCCTTGTATTAGCGCTGGC
PSMT2_R4 CAGTAAATTCACACATTCCGTATCTTCCC
PSMT2_R5 ATTCCCGTTCAAGTAAACATGCGG
PSMT2_R6 ACCGGAATGAGAATGCATAAAGTAAAGG
PSMT3_F1 ATTGTATAGCCAAAGTTGCAGGTAGGG
PSMT3_F2 AGACCGTTTGTACCGAATTCTGC
PSMT3_F3 GCTTCAGCATTGGTTAACGAGTGC
PSMT3
PSMT3 ¨F4 GCAGTGAAAGCCATATCCAAAGC
PSMT3_R1 AACCGTCCCCAAGATGATTCC
PSMT3_R2 TCGTTCCATTCGTGAAGAATGC
PSMT3_R3 GAGGGTAAGCCTCAATAACAGACTGG
PSMT3_R5 TTAAGATCACCAGGCATACTCAAGG
Date Recue/Date Received 2020-06-18

19a
Table 2: Primer combinations used to amplify and sequence PS
Methyltransferases from
genomic DNA
Annealing Extensio
Sequencing primers used for
Methyltransferas Primer
e combination temperature n
time Sanger sequencing of purified
[ C] [5] PCR product
PSMT1 F2/R
¨ 62 60 PSMT1_F2, PSMT1 R2
2
PSMT1 F3/R PSMT1 F3,
¨ 68 60
3 PSMT1_F5,PSMT1_R3
PSMT1 F4/R
PSMT1 ¨ 66 60 PSMT1 F4, PSMT1 R4
4 _ _
PSMT1 F5/R PSMT1_F5, PSMT1 R3,
¨ 63.5 60
PSMT1 _R5
PSMT1 F6/R
¨ 62.8 60 PSMT1_F6, PSMT1 _R1
1
PSMT2_F1, PSMT2_F2,
PSMT2 F1/R PSMT2 F4' PSMT2 F5,
PSMT2 ¨ 60 60 ¨ ¨
6 PSMT2_R1, PSMT2_R4,
PSMT2_R6
PSMT3_F2, PSMT3_F4,
PSMT3 F1/R
PSMT3 ¨ 68.5 60 PSMT3 F5, PSMT3 R1,
5 _ ¨
PSMT3_R2, PSMT3_R3
5 Table 3 Primer combinations used to amplify PS Methyltransferases from
genomic DNA
Methyltransferas Primer Annealing Extensio Expected Figure
e combination temperature n time fragment
size
[ C] [5] [bp]
PSMT1_F1/R 62 60 739 Figure 5a
2
PSMT1_F3/R 66 60 1568 Figure 5b
3
PSMT1 F4/R 68 60 318 Figure Sc
PSMT1 ¨
4
PSMT1_F5/R 63.5 60 1354 Figure 5d
5
PSMT1_F6/R 62.8 60 604 Figure 5e
1
PSMT2_F2/R 60.3 60 613 Figure 10a
3
PSMT2_F4/R 60.9 60 627 Figure 10b
4
PSMT2
PSMT2_F4/R 61.7 60 909 Figure 10c
5
PSMT2_F5/R 62.8 60 664 Figure 10d
6
PSMT3_F1/R 68.5 50 497 Figure 12a
1
PSMT3_F2/R 68.5 50 564 Figure 12b
2
PSMT3
PSMT3_F3/R 68.5 50 511 Figure 12c
3
PSMT3_F4/R 68.5 50 589 Figure 12d
5
Date Recue/Date Received 2020-06-18

19b
Table 4: Primers used to amplify sequences selected for virus induced gene
silencing
Target gene to be silenced Primer name Oligonucleotide sequences (5'- to
3'-)
(in capitals: gene-specific sequence; in
lower case: added sequence; underlined:
restriction sites)
PS PHYTOENE ps_pds_F GAGGTGTTCATTGCCATGTCAA
DESATURASE ps_pds_R4 GTTTCGCAAGCTCCTGCATAGT
VIGS_PSMT1_ aaactcqaqaaqctTGGTCATAATCATCAATC
PS METHYL TRANSFERASE F AG
I VIGS_PSMT1
R
¨ aaaggtaccCATGTACTACTACATCATCTCC
VIGS_PSMT2_ aaactcqaqaaqcttGTGTAACTAAGCCAGCG
PS METHYL TRANSFERASE F C
2 VIGS_PSMT2
R
¨ aaaggtaccACTTGAATATATCACCGC
Materials and Methods
Generation of EST libraries
a) RNA isolation and cDNA synthesis
Material was harvested from stems and capsules at two developmental stages
from four
poppy cultivars. RNA was prepared individually from five plants per cultivar,
developmental
stage and organ. The harvested material was ground in liquid nitrogen using a
mortar and
pestle. RNA was isolated from the ground stem or capsule preparations using a
CTAB
(hexadecyltrimethylammonium bromide) based method as described in Chang et al.
(1993)
Plant Mol. Biol. Rep. 11: 113-116 with slight modifications (three extractions
with
chloroform:isoamylalcohol, RNA precipitation with Lithium chloride at -20 C
over night). RNA
was quantified spectrophotometrically before pooling equal amounts of RNA from
five plants
per cultivar, stage and organ. The pooled samples underwent a final
purification step using
an RNeasy Plus MicroKit (Qiagen, Crawley, UK) to remove any remaining genomic
DNA
from the preparations. RNA was typically eluted in 30-100 pl water. cDNA was
prepared
using a SMART cDNA Library Construction Kit (Clontech, Saint-Germainen-Laye,
France)
according to the manufacturer's instructions but using SuperScript ll Reverse
Transcriptase
(Invitrogen, Paisley, UK) for first strand synthesis. The CDSIII PCR primer
was modified to:
Date Recue/Date Received 2020-06-18

19c
ATT CTA GAT CCR ACA TGT TTT TTT TTT TTT TTT TTT TVN 3' where R = A or G, V =
A, C or G; N = NT or C/G. cDNA was digested with Mmel (New England Biolabs
Inc.,
Hitchin, UK) followed by a final purification using a QIAquick PCR
Purification kit (Qiagen,
Crawley, UK).
5
b) cDNA Pyrosequencing
The Roche 454 GS-FLX sequencing platform (Branford, CT, USA) was used to
perform
pyrosequencing on cDNA samples prepared from the following materials for each
of the
Date Recue/Date Received 2020-06-18

CA 02803102 2012-12-18
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four P. somniferum cultivars GSK MORPHINE CVS2, GSK MORHINE CVS1, GSK
NOSAPINE CVS1 and GSK THEBAINE CVS1:
1. Stem, 1-3 days after petal fall (early harvest)
2. Stem, 4 -6 days after petal fall (late harvest)
5 3. Capsule, 1-3 days after petal fall (early harvest)
4. Capsule, 4 -6 days after petal fall (late harvest)
C) Raw sequence analysis, contiguous sequence assembly and annotation
The raw sequence datasets were derived from parallel tagged sequencing on the
454
10 sequencing platform (Meyer et al. (2008) Nature Protocols 3: 267-278).
Primer and tag
sequences were first removed from all individual sequence reads. Contiguous
sequence
assembly was only performed on sequences longer than 40 nucleotides and
containing
less than 3% unknown (N) residues. These high quality EST sequences were
assembled into unique contiguous sequences with the CAP3 Sequence Assembly
15 Program (Huang and Madan (1999) Genome Res. 9: 868-877), and the
resulting contigs
were annotated locally using the BLAST2 program (Altschul et al. (1997)
Nucleic Acids
Res. 25: 3389-3402) against the non-redundant peptide database downloaded from
the
NCBI.
20 d) Expression profiling of the methyltransferase genes
The number of ESTs associated with the respective methyltransferase consensus
sequences were counted in each of the 16 EST libraries. The values obtained
were
normalised on the basis of total ESTs obtained per library.
RT-PCR on GSK NOSAPINE CVS1 mRNA and direct amplification and sequencing
of full length cDNA of PSMT1.
An aliquot of GSK NOSAPINE CVS1 cDNA prepared for cDNA pyrosequencing (see
above) was used to amplify and sequence the full length coding sequence of
PSMT1
using the following PCR conditions:
Reaction mixture:
5x HF buffer (Finnzymes) 5 I
dNTPs (20 mM each) 0.25 I
Fwd primer (10 M) 2.5 I
Rev primer (10 M) 2.5 I
gDNA (10 ng/ I) 5 I
Phusion Hot Start (Finnzymes) 0.25 I
dH20 9.5 I

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Reaction volume: 25 I
Phusion Hot Start from Finnzymes was purchased through New England Biolabs,
(Bishops Stortford, UK).
PCR programs:
initial denaturation 98 C 1 min
30 cycles of: denaturation 98 C 30 sec
annealing temperature 61 C 30 sec
extension 72 C 40 sec
final extension 72 C 10 min
incubation 4 C storage
primer combination: PSMT1 CLF: ATAGGATCCAACATGGCTACCAATGGCG
PSMT1 CLR: GCGCTCGAGCATTCATTTGTGA
These primers served a dual purpose in that they were used to amplify PSMT1
cDNA for
sequencing and cloning (cloning not described here). The underlined bases of
the
cloning primers delineate the nucleotides which are sequence-specific to the
5' and 3'
end of PSMT1 cDNA, respectively. The PSMT1 specific nucleotide sequence used
to
design the primers were confirmed upon sequencing the PSMT1 gene from genomic
DNA as described below.
PCR products were cleaned with Agencourt AMPure PCR purification kit (Beckman
Coulter, High Wycombe, UK) and sequenced by Sanger sequencing.
Amplification and sequencing of the methyltransferase genes from genomic DNA
a) Genomic DNA preparation
Leaf samples (30-50mg) for DNA extraction were harvested from plants of GSK
MORHINE CVS1, GSK MORPHINE CVS2, GSK NOSAPINE CVS1 and GSK
THEBAINE CVS1 grown in the glasshouse. DNA was extracted using Qiagen
BioSprint
96. Extracted DNA was quantified using Hoescht 33258 and normalized to 10
ng/ul.
b) Amplification and sequencing of the methyltransferase genes from genomic
DNA of
GSK NOSCAPINE CVS1
Primers and primer sequences used for amplification of the respective
methyltransferase
genes from the extracted genomic DNA are shown in Table 1. Primers were
designed
based on the respective methlytransferase contigs assembled from ESTs unique
to

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cultivar GSK NOSCAPINE CVS1. The respective contigs contained the complete
open
reading frame of each methyltransferase as well as 5'and 3' untranslated
regions.
Amplification from genomic DNA was performed using the primer combinations
shown in
Table 2 and the PCR conditions shown below. The PCR amplifications were
performed
on pools of genomic DNA comprising DNA from four individuals. 5 I of each FOR
reaction was resolved on 1.2% Agarose alongside appropriate size standards.
Reaction mixture:
5x HF buffer (Finnzymes) 5 I
dNTPs (20 mM each) 0.25 I
Forward primer (10 M) 2.5 I
Revers primer (10 M) 2.5 I
gDNA (10 ng/ I) 5 I
Phusion Hot Start (Finnzymes) 0.25 I
dH20 9.5 I
Reaction volume: 25 I
.. PCR program:
The annealing temperature and extension time for the respective primer
combinations
are shown in Table 2 and 3.
initial denaturation 98 C 1 min
cycles of: denaturation 98 C 30 sec
25 annealing temperature Table 2&3 30 sec
extension 72 C Table 2 & 3
final extension 72 C 10 min
incubation 4 C storage
The PCR products were purified using the Agencourt AMPure purification kit
(BECKMAN
COULTER UK LTD, Bromley, UK). 30-50 ng of the respective purified FOR product
were
subjected to Sanger-sequencing using the primers indicated in Table 2 as
sequencing
primers.
The amino acid sequences of the respective methyltransferase, deduced from the
open
reading frame sequences confirmed by Sanger-sequencing, were compared to
protein
sequences deposited in the NCBI Non-15 redundant protein database using the
Standard Protein BLAST programme (blastp).
LAnalysis of genomic DNA from GSK NOSAPINE CVS1, GSK MORPHINE CVS2, GSK
MORHINE CVS1 and GSK THEBAINE CVS1 for the presence of the methyltransferase
genes

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To check if the respective methyltransf erase genes were present in all four
cultivars, PCr
amplifications were performed on genomic DNA from GSK NOSAPINE CVS1, GSK
MORPHINE CVS2, GSK MORN INE CVS1 and GSK THEBAINE CVS1 . using the primer
combinations shown in Table 3. Exactly the same PCR conditions as described
above
to amplify the full length genomic sequences from GSK NOSAPINE CVS1 were used
.
The genomic DNA was derived from four individual plants per cultivar. 5 I of
each PCR
reaction was resolved on 1% agarose alongside appropriate size standards.
Analysis of genomic DNA and alkaloid profiling from poppy straw of an F2
population
a) DNA extraction from F2 plants
40-50mg of leaf tissue was harvested, in duplicate, from all poppy plants
within the GSK
NOSCAPINE CVS1 X GSK THEBAINE CVS1 F2 mapping population at the 'small
rosette' growth stage (-10 leaves present on each plant).
Leaf tissue (40 ¨ 50 mg wet weight) was collected into 1.2 ml sample tubes in
8 x 12
format (Part Number 1760-00, Scientific Specialties Inc, 130 Thurman St, Lodi,
CA
95240 USA), closed with strip caps (Part Number 1702-00, Scientific
Specialties Inc) and
shipped to the AGRF (Australian Genome Research Facility) Adelaide on Techni-
Ice dry
Ice packs by overnight courier.
On receipt, strip caps were removed and a 3 mm tungsten carbide bead was added
to
each tube (Part Number 69997, Qiagen GmbH, Hi!den, Germany). Samples were
placed
at -80 C (Freezer model; Sanyo MDF-U73V) for a minimum of two hours prior to
freeze-
drying for 18 hr (Christ Model Alpha 2-4 LSC).
Following freeze drying, tubes were sealed with fresh strip caps (as above),
and samples
were powdered by bead-milling (Model "Tissue Lyser", Part Number 85300;
Qiagen) at
3,000 RPM for 2 x 60 sec cycles separated by plate inversion. DNA extraction
was
performed using the "Nucleospin Plant II" system (Macherey-Nagel, GmbH & Co.
KG
Neumann-Neander-StraBe 6-8, 52355 Duren, Germany).
Cell lysis was performed using the supplied Buffer Set PL2/3. The
manufacturer's
protocol for centrifugal extraction was followed (Centrifuge model 4-K 15;
Sigma
Laborzentrifugen GmbH, 37520 Osterode am Harz, Germany).

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The recovered DNA (12/96 samples, one sample per plate column) was checked for
quality and quantity by ultra violet spectroscopy (Model Nanodrop-8000;
NanoDrop
products, 3411 Silverside Rd, Bancroft Building; Wilmington, DE 19810, USA) at
230,
260 and 280 nM.
b) Genotyping of F2 DNA samples for presence or absence of the methyltransf
erases
DNA samples from a total of 276 F2 plants were genotyped for the presence or
absence
of PSMT1, PSMT2 and PSMT3, respectively, by amplifying a short fragment of
each of
the genes. 5'-VIC-labeled forward primers were used for the amplification of
each of the
methyltransferase fragments, enabling the separation and analysis of the
resulting
fluorescently labeled PCR fragments on the 96-capillary electrophresis 37304
DNA
Analyzer system (Applied Biosystems) according to the manufacturer's
instructions. In
addition to the respective methyltransferase fragments, an internal positive
control was
amplified in each FOR assay in order to distinguish lack of amplification due
to absence
of the repsective methyltransferase genes in a DNA sample from lack of
amplification
due to FOR assay failure.
The following primers were used (primer sequences are shown in Table1):
PSMT1: PSMT1 F3/ PSMT1 R1; amplified fragment size: 129 bp
PSMT2: PSMT2 F4/ PSMT2 R3; amplified fragment size: 284bp
PSMT3: PSMT3 F4/ PSMT3 R2; amplified fragment size: 309 bp
The PSMT1- and PSMT3-fragments were amplified with the following FOR
conditions:
Reaction mixture:
5xGoTaq Buffer (Promega) 2 I
dNTPs (2.5 mM mix) 0.5 I
MgCl2 (25 mM) 0.6 I
Forward primer (10 M) 0.5 I
Revers primer (10 M) 0.5 I
gDNA (5 ng/ I) 2 I
GoTaq (Promega) 0.2 I
dH20 3.7 I
Reaction volume: 10 I
FOR program:
initial denaturation 94 O 1 min
30 cycles of: denaturation 94 O 30 sec
annealing temperature 62 C 30 sec
extension 72 C 20-30 sec
final extension 72 C 5 min

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incubation 4 C storage
The PSMT2- fragment was amplified with the following PCR conditions:
5 Reaction mixture:
5xType-it multiplex PCR mix (Qiagen) 5 I
Forward primer (10 M) 0.5 I
Revers primer (10 M) 0.5 I
10 gDNA (5 ng/ I) 2 I
dH20 2 I
Reaction volume: 10 I
15 PCR program:
initial denaturation 95 C 15 min
cycles of: denaturation 95 C 15 sec
annealing temperature 60 C 30 sec
20 extension 72 C 30 sec
final extension 72 C 5 min
incubation 4 C storage
c) Poppy straw analysis from F2 plants
25 Poppy capsules were harvested by hand from the mapping population once
capsules
had dried to approximately 10% moisture on the plant. The seed was manually
separated from the capsule, and capsule straw material (Poppy Straw) was then
shipped
to the GSK extraction facility in Port Fairy, Australia.
30 The poppy straw samples were then ground in a Retsch Model MMO4 ball
mill into a fine
powder. Two gram samples of ground poppy straw were then weighed accurately (2

0.003 g) and extracted in 50mL of a 10% acetic acid solution. The extraction
suspension
was shaken on an orbital shaker at 200rpm for a minimum of 10 minutes then
filtered to
provide a clear filtrate. The final filtrate was passed through a 0.22 pm
filter prior to
analysis.
The solutions were analysed using a Waters Acquity UPLC system fitted with a
Waters
Acquity BEH C18 column, 2.1mm x 100mm with 1.7 micron packing. The mobile
phase
used a gradient profile with eluent A consisting of 0.1% Tetrafluoroacetic
acid in
deionised water and eluent B consisting of 100% Acetonitrile. The mobile phase
gradient
conditions used are as listed in Table 2, the gradient curve number as
determined using
a Waters Empower chromatography software package. The flow rate was 0.6mL per

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WO 2011/161431 PCT/GB2011/051121
minute and the column maintained at 450. The injection volume was 14 injection
volume and the alkaloids were detected using a UV detector at 285nm.
The loss on drying (LOD) of the straw was determined by drying in an oven at
105
degrees centrigrade for 3 hours.
- Gradient Flow Program:
% Eluent Flow
TIME (minutes) % Eluent A Curve No
(mL/min)
0.00 95.0 5.0 0.60 INITIAL
0.80 90.0 10.0 0.60 6
3.40 75.0 25.0 0.60 3
3.60 95.0 5.0 0.60 6
4.00 95.0 5.0 0.60 11
Alkaloid concentrations for morphine, codeine, thebaine, oripavine and
noscapine were
determined by comparison with standard solutions and the results calculated on
a dry
weight basis.
Typical retention times are as follows:
Compound Retention Time (minutes)
Morphine 1.14
Pseudo morphine 1.26
Codeine 1.69
Oripavine 1.80
10-Hydroxythebaine 2.32
Thebaine 2.53
Noscapine 3.16
Virus induced gene silencing (VIGS) of PSMT1 and PSMT2
a) Generation of silencing constructs
A tobacco rattle virus (TRV) based virus induced gene silencing system
developed and
described by Liu et al. (2002) Plant J. 30(4): 415-429 was used to investigate
the gene

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function of PSMT1 and PSMT2. DNA fragments selected for silencing of PSMT1 and
PSMT2, respectively, were amplified by PCR and cloned into the silencing
vector pTRV2
(GenBank accession no. AF406991; Liu et al. (2002) Plant J. 30(4): 415-429).
They
were linked to a 129 bp-long fragment of the P. somniferum Phytoene desaturase
gene
(PsPDS) in order to silence the respective methylatransferases and PsPDS
simultaneously. Plants displaying the photo-bleaching phenotype that resulted
from
silencing of PsPDS (Hileman et al. (2005) Plant J. 44(2): 334-341) were
identified as
plants successfully infected with the respective silencing constructs and
selected for
analysis.
Generation of the pTRV2-PDS construct: A 622 bp fragment (Figure 13) of PsPDS
was
amplified from cDNA prepared from GSK NOSCAPINE CVS1 as described above using
primers ps pds F and ps_pds R4 (Table 4). The sequence of the forward primer
was
based on a 412 bp long contig derived from the EST-libraries which shared 99%
identity
at its 3' end with the partial coding sequence of the P. somniferum phytoene
desaturase
(GenBank accession no. DQ116056). The sequence of the reverse primer was
designed based on the DQ116056 sequence. The PCR conditions were identical to
those described above for the amplification of PSMT1 from genomic sequence
except
that the annealing step was carried out at 70 C and the extension time was
increased to
60 seconds.
Sau3A1 digestion of the PCR-fragment yielded among others two fragments (280
bp and
129 bp in length) that carried BamH1-compatible sticky ends at both, their 5'
and 3' ends.
The 129 bp long fragment (underlined stretch in Figure 13) was cloned into the
BamHI
site of the pTRV2 vector. Because Sau3A1 was used to produce BamHI-compatible
sticky ends, the BamHI site at the 5-end of the PDS-insert was abolished in
the pYL156-
PDS construct. However, the BamHI recognition site at its 3'-end was kept
intact due to
the nature of the PDS-insert sequence.
A sequence-confirmed pTRV2-PDS construct, with the 129 bp fragment in sense
orientation, was subsequently used as a vector for generating the PSMT1 and
PSMT2
silencing constructs, and served as a control in the VIGS experiments.
Generation of silencing constructs for PS PSMT1 and PSMT2 (pTRV2-PDS-PSMT1 and
pTRV2-PDS-PSMT2): The DNA fragments selected for silencing PSSOMT1and
PSSOMT2 were amplified from cDNA of GSK NOSCAPINE CVS1 prepared as

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described above with the use of the primer sequences shown in Table 5.
Additional
restriction sites (forward primers: Xhol and HindlIl for forward primers; Kpnl
site for
reverse primers) were added to the gene-specific primers in order to
facilitate cloning.
The amplification conditions were as described above for amplifying the PDS-
fragment
.. except that the annealing temperatures were 60.9 C for PSMT1and 62.8 C for
PSMT2
and the extension time was 30 seconds.
The sequence selected to silence PSMT1 (Figure 14) was cloned into pTV00
(Ratcliff et
al. (2001) Plant J. 25(2): 237-245) using HindlIl and Kpnl and subcloned into
pTRV2-
PDS using BamHI and Kpnl. A sequence-confirmed pTRV2-PDS-PSMT1 construct was
used in the VIGS experiments.
The sequence selected for silencing PSMT2 (Figure 15) was cloned directly into
pTRV2-
PDS. Since the PCR amplification was carried out with Phusion (Finnzymes,
Finland)
the resulting PCR fragment carried blunt ends and, after cutting with Xhol,
was cloned
into pYL156-PDS cut with Xhol and Smal. A sequence-confirmed pTRV2-PDS-PSMT2
construct was used in the VIGS experiments.
b) Transformation of constructs into Agrobacterium tumefaciens
.. The propagation of the silencing constructs was carried out with the E.
coli strain DH5a
and, subsequently, the respective silencing constructs, as well as pTRV1
(GenBank
accession no. AF406990; Liu et al. (2002) Plant J. 30(4): 415-429) were
independently
transformed into electrocompetent Agrobacterium tumefaciens (strain GV3101).
c) Infiltration of plants
Overnight liquid cultures of A. tumefaciens containing each silencing
construct were
used to inoculate Luria-Bertani (LB) medium containing 10 mM MES, 20 pM
acetosyringone and 50pg/mIkanannycin. Cultures were maintained at 28 C for 24
hours,
harvested by centrifugation at 3000g for 20 min, and resuspended in
infiltration solution
(10 mM MES, 200 pM acetosyringone, 10 mM MgCl2) to an 0D600 of 2.5. A.
tumefaciens harbouring the respective constructs (pTRV2-PDS-PSMT1, pTRV2-PDS-
PSMT2 or, as a control, pTRV2-PDS) were each mixed 1:1 (v/v) with A.
tumefaciens
containing pTRV1, and incubated for two hours at 22 C prior to infiltration.
Two weeks
old seedlings of GSK NOSCAPINE CVS1 grown under standard greenhouse conditions
(22 C, 16h photoperiod), with emerging first leaves, were infiltrated as
described by
Nagel and Facchini (2010) Nat. Chem. Biol. 6: 273-275.

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d) Latex and capsule analysis of silenced plants
Leaf latex of infiltrated opium poppy plants displaying photo-bleaching as a
visual marker
for successful infection and silencing was analysed when the first flower buds
emerged
(-7 week old plants). Plants showing a similar degree of photo-bleaching of
leaves were
selected for analysis.
Latex was collected from cut petioles, with a single drop dispersed into 500 4
10%
acetic acid. This was diluted 10x in 1% acetic acid to give an alkaloid
solution in 2%
acetic acid for further analysis. Capsules were harvested by hand from
glasshouse-
grown from the same plants used for latex analysis and single capsules were
ground in
a Retsch Model MMO4 ball mill into a fine powder. Ten mg samples of ground
poppy
straw were then weighed accurately (10 0.1 mg) and extracted in 0.5 mL of a
10%
acetic acid solution with gentle shaking for lh at room temperature. Samples
were then
clarified by centrifugation and a 50 4 subsample diluted 10x in 1% acetic acid
to give an
alkaloid solution in 2% acetic acid for further analysis.
All solutions were analysed using a Waters Acquity UPLC system fitted with a
Waters
Acquity BEH 018 column, 2.1mm x 100mm with 1.7 micron packing. The mobile
phase
used a gradient profile with eluent A consisting of 10mM ammonium bicarbonate
pH
10.2 and eluent B methanol. The mobile phase gradient conditions used are as
listed in
Table 1, with a linear gradient. The flow rate was 0.5mL per minute and the
column
maintained at 60 C. The injection volume was 24 and eluted peaks were ionised
in
positive APCI mode and detected within -3 ppm mass accuracy using a Thermo LTQ-
Orbitrap. The runs were controlled by Thermo Xcalibur software.
- Gradient Flow Program:
% Eluent Flow
TIME (minutes) % Eluent A
B (mL/min)
0.0 98.0 2.0 0.50
0.2 98.0 2.0 0.50
0.5 60.0 40 0.50
4.0 20.0 80.0 0.50
4.5 20.0 0.0 0.50
All data analysis was carried out in R. Putative alkaloid peaks were
quantified by their
pseudomolecular ion areas using custom scripts. Peak lists were compiled and
any

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peak-wise significant differences between samples were identified using 1-way
ANOVA
with p-values adjusted using the Bonferroni correction for the number of
unique peaks in
the data set. For any peak-wise comparisons with adjusted p-values < 0.05,
Tukey's
HSD test was used to identify peaks that were significantly different between
any given
5 sample and the control. Alkaloids were identified by comparing exact mass
and
retention time values to those of standards. Where standards were not
available,
neutral exact masses were used to generate molecular formulae hits within
elemental
constraints of C = 1:100, H = 1:200, 0 = 0:200, N = 0:3 and mass accuracy <
20ppm.
The hit with the lowest ppm error within these constraints was used to assign
a putative
10 formula.
Example 1
Assembly of full length PSMT1 cDNA sequence from ESTs and confirmation by
15 sequencing of full length cDNA
The full length cDNA sequence for PSMT1 (Figure la) was assembled from ESTs
derived from the 454 sequencing platform using the CAP3 sequence assembly
programme. The full length cDNA sequence was confirmed by RT-PCR and direct
20 amplification of the full length cDNA from GSK NOSAPINE CVS1 mRNA.
Example 2
PSMT1 is exclusively expressed in the noscapine producing Papaver somniferum
cultivar GSK NOSAPINE CVS1.
Figure 2 shows the normalized distribution of ESTs associated with the PSMT1
consensus sequence across each of the 16 EST libraries prepared from two
organs
(capsules and stems) at two developmental stages (early and late harvest) from
each of
the four poppy cultivars, GSK MORHINE CVS1, GSK MORPHINE CVS2, GSK
NOSAPINE CVS1 and GSK THEBAINE CVS1. ESTs corresponding to PsSOMT were
exclusively found in libraries derived from the noscapine producing cultivar
GSK
NOSAPINE CVS1 (Figure 2). PSMT1 expression was strongest in stem tissue
shortly
after flowering.

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Example 3
PCR-amplification of PSMT1 from genomic DNA of the four Popover somniferum
cultivars GSK NOSAPINE CVS1, GSK MORHINE CVS1, GSK MORPHINE CVS2 and
GSK THEBAINE CVS1.
PCR-amplifications of PSMT1 fragments were performed on genomic DNA from the
four
poppy cultivars GSK NOSAPINE CVS1 (noscapine cultivar), GSK MORHINE CVS1 and
GSK MORPHINE CVS2 (morphine cultivars) and GSK THEBAINE CVS1 (thebaine
cultivar) using the primer combinations shown in Table 2 and 3.
Figure 5 shows the PCR-amplification of PSMT1 from genomic DNA of the four
Papaver somniferum cultivars GSK NOSAPINE CVS1, GSK MORHINE CVS1, GSK
MORPHINE CVS2 and GSK THEBAINE CVS1.
The amplification from genomic DNA yielded the gene sequence shown in Figure
3a.
Example 4
The putative protein encoded by PSMT1 shows highest sequence similarity to (S)-
scoulerine 9-0-methyltransferases from Coptis japonica and Thalictrum flavum
The closest homologues to the putative protein encoded by the PSMT1 open
reading
frame (Figure 4) are (S)-scoulerine 9-0-methyltransferases from Coptis
japonica
(Accession: 039522.1, Evalue: le-120, 61% identical) and from Thalictrum
flavum
(Accession: AAU20770.1, Evalue: 5e-118 59% identical).
Example 5
PSMT1 is only present in the genome of the noscapine producing P. somniferum
cultivar GSK NOSAPINE CVS1
The transcribed region covered by the ESTs contained the complete coding
sequence of
PSMT1 (including 5' and 3' untranslated regions), which was used for primer
design
(Table 1) to amplify the PSMT1 gene from genomic DNA in a series of
overlapping
fragments for sequencing (Figure 3a). Upon testing a subset of the primer
combinations
(Table 3) on genomic DNA samples from all four cultivars it was discovered
that the
PSMT1 fragments could only be amplified from genomic DNA of the noscapine
producing cultivar GSK NOSAPINE CVS1 but not from genomic DNA of the

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predominantly morphine (GSK MORHINE CVS1 and GSK MORPHINE CVS2) or
thebaine (GSK THEBAINE CVS1) producing cultivars (Figure 5). The PCR
amplifications
were performed on pools of genomic DNA comprising DNA from four individuals
per
cultivar. This discovery explains why the PSMT1 is only expressed in the GSK
NOSAPINE CVS1 cultivar and is absent from the transcriptome of the other three
cultivars.
Example 6
Assembly of full length PSMT2 cDNA sequence from ESTs and confirmation by
sequencing from genomic DNA
The full length cDNA sequence for PSMT2 (Figure lb) was assembled from ESTs
derived from the 454 sequencing platform using the CAP3 sequence assembly
programme. The full length cDNA sequence was confirmed by amplification and
sequencing from genomic DNA.
Example 7
PSMT2 is exclusively expressed in the noscapine producing Popover somniferum
cultivar GSK NOSCAPINE CVS1
Figure 9 shows the normalized distribution of ESTs associated with the PSMT2
consensus sequence across each of the 16 EST libraries prepared from two
organs
(capsules and stems) at two developmental stages (early and late harvest) from
each of
the four poppy cultivars, GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK
NOSCAPINE CVS1 and GSK THEBAINE CVS1. ESTs corresponding to PSMT2 were
exclusively found in libraries derived from the noscapine producing cultivar
GSK
NOSCAPINE CVS1 . PSMT2 expression was strongest in stem tissue shortly after
flowering.
Example 8
PCR-amplification of PSMT2 from genomic DNA of the four Papa ver somniferum
cultivars GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK NOSCAPINE CVS1
and GSK THEBAINE CVS1.
PCR-amplifications of PSMT2 fragments were performed on genomic DNA from the
four poppy cultivars GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK

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WO 2011/161431 PCT/GB2011/051121
NOSCAPINE CVS1 and GSK THEBAINE CVS1 using the primer combinations shown in
Table 2 and 3.
Figure 10 shows the PCR-amplification of PSMT2 from genomic DNA of the four
Papaver somniferum cultivars GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK
NOSCAPINE CVS1 and GSK THEBAINE CVS1;
The amplification from genomic DNA yielded the gene sequence shown in Figure
3b.
Example 9
The putative protein encoded by PSMT2 shows highest sequence similarity to
norcoclaurine 6-0-methyltransferases from Coptis japonica, Papa ver bra
cteatum
and Papa ver somniferum
The closest homologues to the putative protein encoded by the PSMT2 open
reading
frame (Figure 1 b) are (R,S)-norcoclaurine 6-0-methyltransferase from Coptis
japonica
(accession: Q9LEL6, identities: 146/355 (42%)), putative norcoclaurine 6-0-
methyltransferase from Papaver bracteatum (accession: AC090232, identitities:
151/346
(44%)) and S-adenosyl-L-methionine:norcoclaurine 6-0-methyltransferase from
Papa ver
somniferum (accession: AAP45315, identities = 148/343 (44%)). The sequence
comparisons were carried out using NCBI's 'blastp' algorithm (method:
compositional
matrix adjust).
Example 10
PSMT2 is only present in the genome of the noscapine producing P. somniferum
cultivar GSK NOSCAPINE CVS1.
The transcribed region covered by the ESTs contained the complete coding
sequence of
.. PSMT2 (including 5' and 3' untranslated regions), which was used for primer
design
(Table 1) to amplify the PSMT2 gene from genomic DNA in a series of
overlapping
fragments for sequencing (Figure 3b). Upon testing a subset of the primer
combinations
on genomic DNA samples from all four cultivars it was discovered that the
PsSOMT2
fragments could only be amplified from genomic DNA of the noscapine producing
cultivar GSK NOSCAPINE CVS1 but not from genomic DNA of the predominantly
morphine (GSK MORPHINE CVS1, GSK MORPHINE) or thebaine (GSK THEBAINE

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WO 2011/161431 PCT/GB2011/051121
CVS1) producing cultivars (Figure 10). The PCR amplifications were performed
on pools
of genomic DNA comprising DNA from four individuals per cultivar using primer
combinations shown in Table 3. This discovery explains why the PSMT2 is only
expressed in the GSK NOSCAPINE CVS1 cultivar and is absent from the
transcriptome
of the other three cultivars.
Example 11
Assembly of full length PSMT3 cDNA sequence from ESTs and confirmation by
sequencing from genomic DNA
The full length cDNA sequence for PSMT3 (Figure 1c)was assembled from ESTs
derived from the 454 sequencing platform using the CAP3 sequence assembly
programme. The full length cDNA sequence was confirmed by amplification and
sequencing from genomic DNA.
Example 12
PSMT3 is exclusively expressed in the noscapine producing Papaver somniferum
cultivar GSK NOSCAPINE CVS1.
Figure 11 shows the normalized distribution of ESTs associated with the PSMT3
consensus sequence across each of the 16 EST libraries prepared from two
organs
(capsules and stems) at two developmental stages (early and late harvest) from
each of
the four poppy cultivars, GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK
NOSCAPINE CVS1 and GSK THEBAINE CVS1. ESTs corresponding to PsSOMT3
were exclusively found in libraries derived from the noscapine producing
cultivar GSK
NOSCAPINE CVS1 (Figure 11). PSMT3 expression was strongest in stem tissue
shortly
after flowering.
Example 13
PCR-amplification of PSMT3 from genomic DNA of the four Papa ver somniferum
cultivars GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK NOSCAPINE CVS1
and GSK THEBAINE CVS1.
PCR-amplifications of PSMT3 fragments were performed on genomic DNA from the
four
poppy cultivars GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK NOSCAPINE

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CVS1 and GSK THEBAINE CVS1 using the primer combinations shown in Table 2 and
3.
Figure 12 shows the PCR-amplification of PSMT3 from genomic DNA of the four
5 Papaver somniferum cultivars GSK MORPHINE CVS1, GSK MORPHINE CVS2, GSK
NOSCAPINE CVS1 and GSK THEBAINE CVS1;
The amplification from genomic DNA yielded the gene sequence shown in Figure
3c.
10 Example 14
The putative protein encoded by PSMT3 shows highest sequence similarity to
norcoclaurine 6-0-methyltransferases from Papaver somniferum and Papaver
bracteatum
15 The closest homologues to the putative protein encoded by the PSMT3 open
reading
frame (Figure 4) are (R,S)-norcoclaurine 6-0-methyltransferases from Papaver
somniferum (accession: AAQ01669, identities: 275/346 (80%), Papaver somniferum
(accession: AAP45315, identities = 275/346 (80%), and Papaver bracteatum
(accession:
AC090232, identities: 274/346 (80%)). The sequence comparisons were carried
out
20 using NCBI's `blastp' algorithm (method: compositional matrix adjust).
Example 15
PSMT3 is only present in the genome of the noscapine producing P. somniferum
25 cultivar GSK NOSCAPINE CVS1.
The transcribed region covered by the ESTs contained the complete coding
sequence of
PSMT3 (including 5' and 3' untranslated regions), which was used for primer
design
(Table 1) to amplify the PSMT3 gene from genomic DNA in a series of
overlapping
30 fragments for sequencing (Figure 3c). Upon testing a subset of the
primer combinations
on genomic DNA samples from all four cultivars it was discovered that the
PSMT3
fragments could only be amplified from genomic DNA of the noscapine producing
cultivar GSK NOSCAPINE CVS1 but not from genomic DNA of the predominantly
morphine (GSK MORPHINE CVS1, GSK MORPHINE) or thebaine (GSK THEBAINE
35 CVS1) producing cultivars (Figure 12). The FOR amplifications were
performed on pools
of genomic DNA comprising DNA from four individuals per cultivar using primer

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WO 2011/161431 PCT/GB2011/051121
combinations shown in Table 3. This discovery explains why the PSMT3 is only
expressed in the GSK NOSCAPINE CVS1 cultivar and is absent from the
transcriptome
of the other three cultivars.
Example 16
Scoulerine and putative narcotoline accumulate in PSMT1- and PSMT2-silenced
plants,
respectively. Virus induced gene silencing led to the accumulation of
scoulerine in both
latex and mature capsules of PSMT1-silenced plants but not of PSMT2-silenced
plants,
PDS-silenced control plants or uninfected plants of GSK NOSCAPINE CVS1 (Figure
16). Scoulerine has been shown to be an intermediate in the biosynthesis of
noscapine
(Battersby et al. (1968) J. Chem. Soc. (C): 2163-2172).
Putative narcotoline (4-desmethyl-noscapine) accumulated in latex and capsules
of
PSMT2-silenced plants but not of PSMT1-silenced plants, PDS-silenced control
plants or
.. uninfected plants of GSK NOSCAPINE CVS1 (Figure 17).
Example 17
PSMT1, PSMT2 and PSMT3 are linked and involved in noscapine biosynthesis
All three methyltransf erase genes are present in all F2 plants that do
contain noscapine.
All F2 plants that do not contain the methlytransferase genes do not make
noscapine
(Figure 6a-c).
30

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